The present invention relates to means for improving the flavour of cheeses and of cheese specialities.
Pressed cheeses without surface flora, which are manufactured essentially with lactococci, have little flavour; its development requires very long ripening times, of the order of 3 to 6 months or even more, for cheeses of the Gouda or Cheddar type, whereas most of these pastes are marketed after considerably shorter ripening times (of the order of a few weeks).
One of the principle objectives of cheese manufacturers is to enhance the flavour of these cheeses, without substantially modifying their manufacturing technology, and without extending the ripening time.
The enzymatic degradation of amino acids is one of the routes of production of flavour molecules. Indeed, amino acids, and in particular aromatic amino acids, branched amino acids and sulphur-containing amino acids are precursors of flavour compounds of the aldehyde, alcohol, acid or thiol type. Some of these compounds have been identified in cheeses and participate in their flavour [(DUMONT et al., Lait 54:31-43, (1974); MC CUGAN, J. Agric. Food Chem. 23:1047-1050, (1975), GREEN and MANNING, J. Dairy Res. 49:737-748, (19382), NEY and WIROTAMA, Z. Lebensm. -Unters, -Forsch. 146:337-343, (1971)].
It has therefore been proposed to enhance the proteolysis in cheeses so as to increase the quantity of free amino acids. The proteolytic system of lactococci has been widely studied, several peptidases have been purified and characterized and their genes have been cloned and sequenced [LAW and MULHOLLAND, Int. Diary J. 5:833-854, (1995)]. Genetically-modified strains which overexpress these peptidases have been constructed; the use of such strains for the manufacture of xe2x80x9cCheddarxe2x80x9d type cheeses was recently described [MC GARRY et al., Appl. Environ. Microbiol. 60:4226-4233, (1994), CHRISTENSEN et al., Int. Dairy J. 5:367-379, (1995)]. However, although the over-expression of peptidases increases the accumulation of the free amino acids, it does not significantly affect the development of flavour. It therefore appears that the factors limiting the development of flavour do not exist at the level of the production of free amino acids, but are also involved in their degradation.
Activities for converting amino acids into flavour compounds nevertheless exist in lactococci. ENGELS and VISSER, [Neth. Milk Dairy J. 50:3-17, (1996)] have shown that flavours typical of Gouda could be generated by incubating cellular extracts of lactococci with methionine. The enzyme assumed to be responsible for this conversion has been purified and characterized; it is a cystathionine xcex2-lyase [ALTING et al., Appl. Environ. Microbiol. 61:4037-4042, (1995)].
It has also been observed that lactococci were capable of degrading in vitro aromatic amino acids and branched amino acids into flavour compounds of the hydroxy acid and acid type. The first step of the degradation of these amino acids is a transamination which requires the presence of an acceptor keto acid [THIROUIN et al., abstr. M4, Club des Bactxc3xa9ries Lactiques (Lactic Acid Bacteria Club)xe2x80x947th colloquium, Paris, France (1995)]. The transamination is also involved in the degradation of methionine to methanethiol [ALTING et al., Fifth symposium on Lactic acid bacteria: Genetics, metabolism and applications, Veldhoven, The Netherland, Sep. 8-12 1996].
Using a strain of L. lactis ssp cremoris, the Inventors"" team has purified and characterized an amino transferase, and observed that in a simple liquid medium containing glucose, this enzyme could, in the presence of xcex1-ketoglutarate, catalyse the transamination of the three aromatic amino acids (phenylalanine, tryptophan and tyrosine), of leucine and of methionine; this enzyme is active under temperature, pH and ionic strength conditions similar to those encountered during the refining of cheese [YVON et al., Appl. Env. Microbiol., 63, 414-419 (1997)]. The Inventors"" team has moreover identified two other aminotransferases which are active on branched amino acids, and which use xcex1-ketoglutarate and, to a lesser degree, oxaloacetate as amino group acceptor. These keto acids may come from the degradation of glutamate or of aspartate, which are always present in cheeses in a large quantity, or they may be synthesized from acetyl-CoA, since the portion of the Krebs cycle between oxaloacetate and xcex1-ketoglutarate appears to be operational in lactococci [LOUBIERE et al., Le Lait 76 (1-2): 5-12, (1996)].
However, the Inventors have observed that in experimental cheeses manufactured with the abovementioned strain of L. lactis ssp cremoris, the degradation of the aromatic amino acids was in fact very low (2 to 5%) which suggested the existence of factors limiting this degradation in cheeses. Among these limiting factors, the most probable existed at the level of the diffusion of the amino acids and their transport inside the energetically drained bacteria cells (because of the fact that at the time of refining, practically all the sugars which can be used as energy source have already been consumed).
However, the Inventors made the hypothesis that the quantity of acceptor keto acids present in cheese represented the first limiting factor.
To verify this hypothesis, the Inventors have, in the first instance, studied, in simple liquid media, the effect of the addition of simple xcex1-ketoglutarate and of oxaloacetate on the degradation of aromatic and branched amino acids by whole lactococci cells. They have thus observed that the addition of xcex1-ketoglutarate increased the degradation of the aromatic and branched amino acids, and that the addition of oxaloacetate increased that of the branched amino acids. They then sought to confirm this result in cheeses, and observed that in this case, only the addition of xcex1-ketoglutarate had an action on the degradation of the amino acids.
The present invention relates to the use of keto acids and in particular of xcex1-ketoglutarate as preparation additive, for enhancing the flavour of a cheese or of a cheese-flavoured food product whose preparation comprises a step of maturation (ripening) in the presence of lactic acid bacteria, and in particular of lactococci. This enhancement of the flavour results from the increase in the catabolism of the amino acids by the said bacteria.
The present invention may be used within the framework of the manufacture of various types of cheese. It is particularly advantageous for the manufacture of pressed cheeses without surface flora, in particular of cheeses with artificial rind. It may also be used within the framework of the manufacture of food products on which it is desired to confer a cheese flavour, among which there may be mentioned in particular food produces in which at least one of the ingredients is obtained from a curd or a milk protein concentrate (casein+whey proteins), such as enzyme-modified cheese bases ( less than  less than Enzyme Modified Cheese  greater than  greater than ), cheese specialities, processed cheeses, low-fat cheeses.
The subject of the invention is in particular a process for the production of a cheese or of a cheese-flavoured food product, characterized in that a preparation additive comprising at least one keto acid chosen from the group consisting of xcex1-ketoglutarate, and the keto acids which are direct precursors of flavour compounds, such as xcex1-ketoisocaproate, ketoisovalerate, and phenylpyruvate, is used to enhance the flavour of the said product.
According to a preferred embodiment of the present invention, the preparation of the said product comprises a maturation step in the presence of at least one lactic acid bacterium belonging to one of the genera Lactococcus, Lactobacillus, Streptococcus and Leuconostoc, and the said additive is added to the said product, prior to the said maturation step or during it.
According to a preferred embodiment of the present invention, the said lactic acid bacterium is chosen from the group consisting of Lactococcus lactis ssp lactis, Lactococcus lactis ssp. cremoris, Lactococcus lactis ssp. diacetylactis, Lactobacllus delbrueckii lactis, Lactobacillus delbrueckii bulgaricus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus paracasei, Streptococcus thermophilus. 
When the said additive comprises xcex1-ketoglutarate, the transamination reaction produces, on the one hand, precursors of flavour compounds and, on the other hand, glutamate, which is in fact a taste enhancer. When it comprises keto acids which are direct precursors of flavour compounds, these may, like xcex1-ketoglutarate, play a role of acceptor for the transamination reactions, and may also be directly degraded into various flavour compounds, which makes it possible to obtain different tastes depending on the keto acid(s) chosen.
The quantity of additive which is used may vary according to the degree of flavour enhancement which it is desired to obtain. For example, xcex1-ketoglutarate may generally be used in an amount of 0.5 to 10 mg per gram of non-matured product (non-matured product is understood to mean the drained curd, or the ultrafiltration retentate, in the case of products obtained by ultrafiltration of milk).
To carry out the invention, the said additive may be introduced directly or indirectly into the product during manufacture. The direct introduction may be easily carried out, for example, by soaking the non-matured product or during maturation, in a solution of xcex1-ketoglutarate, or by impregnating the product with a concentrated solution of xcex1-ketoglutarate, before or after salting, or by adding the keto acid(s) to the salt or to the brine used for the salting, or at the time of lactose removal in the case of lactose-free cheeses, or alternatively, in the case of the products obtained by ultrafiltration, by adding to the ultrafiltration retentate.
Advanatageously, when the xcex1-ketoglutarate is introduced into the brine used for the salting, it is added thereto in an amount of 10 to 100 grams per liter of brine.
The indirect introduction may be made by the addition of a strain capable of producing xcex1-ketoglutarate from the glutamate present in cheeses. This may be either a strain of lactococcus or of another lactic acid bacterium, or another refining microorganism, which strain may have been selected or genetically modified.
The subject of the present invention also covers the cheeses capable of being obtained by the process in accordance with the invention.
The present invention will be understood more clearly with the aid of the additional description which follows, which refers to nonlimiting examples showing the influence of the addition of a keto acid on the catabolism of amino acids by lactococci, and of the addition of xcex1-ketoglutarate to cheeses on their organoleptic qualities, and on the degradation of the amino acids in these cheeses.